Taken together, the initial additions of CuI to each domain caused the disappearance of a large set of NOESY cross-peaks and the parallel appearance of another set of cross-peaks, until
Trang 1of vertebrate Zn-metallothionein-1, respectively, induces significant structural changes
Benedikt Dolderer1, Hartmut Echner1, Alexander Beck1, Hans-Ju¨rgen Hartmann1, Ulrich Weser1, Claudio Luchinat2and Cristina Del Bianco2
1 Interfakulta¨res Institut fu¨r Biochemie, University of Tu¨bingen, Germany
2 Magnetic Resonance Center, University of Florence, Sesto Fiorentino, Italy
The first member of the metallothionein (MT) family
was isolated in 1957 [1] Since then, a large number
of proteins have been described featuring common
characteristics They include ubiquitous small
cys-teine-rich proteins (50–70 amino acids) that are able
to bind many d10metal ions [2] A wealth of different
biological functions has been proposed and continues
to be discovered Obviously, MTs play important
roles in minimizing the uncontrolled reactions of
heavy metal ions like cadmium and the homeostasis
of essential metal ions including copper(I) and zinc(II)
ions [2,3] They are known to successfully cope with
oxidative stress and ionizing radiation [4,5] Other
functions may be associated with the occurrence of tissue-specific isoforms, such as the brain-specific iso-form MT-3, which acts as neuronal growth inhibitory factor [6,7]
Both mammalian MT-1 and MT-2 are composed of the N-terminal b- and the C-terminal a-domain They are predominantly isolated containing zinc or cadmium exclusively bound to cysteine thiolates The nine cys-teine residues of the b-domain accommodate a metal (M)(II)3S9 cluster, while 11 cysteine residues contribute
to the formation of a M(II)4S11cluster in the a-domain [8] However, under certain physiological conditions, e.g when isolated from fetal liver, mammalian MT-1
Keywords
copper; domain; metallothionein; protein
structure; 2D NMR
Correspondence
U Weser, Anorganische Biochemie,
Interfakulta¨res Institut fu¨r Biochemie,
University of Tu¨bingen, Hoppe-Seyler-Str 4,
D-72076 Tu¨bingen, Germany
Fax ⁄ Tel: +49 7071295564
E-mail: ulrich.weser@uni-tuebingen.de
(Received 17 January 2007, revised 28
February 2007, accepted 5 March 2007)
doi:10.1111/j.1742-4658.2007.05770.x
Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d10-metal control To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1H NMR using two synthetic fragments representing the a- and the b-domain The 1H NMR data and thus the structures of Zn4a metallo-thionein (MT)-1 and Zn3bMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1 Cu(I) titra-tion of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing a- and the four Cu(I)-con-taining b-domains The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes Further addi-tion of Cu(I) to the two single domains led to the loss of defined domain structures Upon mixing of the separately prepared aqueous three and four Cu(I) loaded a- and b-domains, no interaction was seen between the two species There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains
Abbreviations
M, metal; MT, metallothionein.
Trang 2and MT-2 are also found to be enriched with Cu(I)
[9] For other members of the MT family, different
metal cluster architectures were reported The
previ-ously mentioned MT-3, which is also a two-domain
protein, for example, is composed of a Cu(I)4S9 cluster
in the N-terminal b-domain and a Zn(II)4S11cluster in
the C-terminal a-domain [10,11] Examples for solely
Cu(I) binding MTs are Cu(I)8 thionein from
Saccharo-myces cerevisiae and Cu(I)6 thionein from Neurospora
crassa [12–14] Differently from other described MTs,
these two fungal proteins consist only of a single
domain harbouring homometallic Cu(I) thiolate
clus-ters [13,14]
The three-dimensional structure of Cd5Zn2MT-2,
isolated from rat liver after cadmium
supplementa-tion, was determined using both NMR and X-ray
crystallography [15] The entire protein is
dumbbell-shaped and contains two independent domains The
polypeptide backbone wraps around the metal
thio-late core forming the scaffold of the two domains
All metal ions are tetrahedrally surrounded by four
thiolate sulphur atoms In the N-terminal b-domain,
the three metal ions and the three bridging thiolate
sulphurs are ordered to form a distorted chair The
C-terminal a-domain is characterized by an
adaman-tane-like four-metal cluster Solution structures of
113Cd-substituted Cd7MT-2 from rabbit, rat and
human are available and revealed structural identity
with the structure of Cd5Zn2MT-2 [8] Comparative
NMR studies provided evidence that Zn(II) can
iso-morphically replace Cd(II) in MT-2 [16] This result
was corroborated by NMR studies on cobalt
substi-tuted MTs, as cobalt was often used as a zinc
ana-logue in structural investigations [17–19] The solution
structure of murine 113Cd7MT-1 showed high
similar-ity with rat liver MT-2 Its b-domain, however,
turned out to be more flexible than in the latter
protein, exhibiting enhanced cadmium–cadmium
exchange rates [20]
The structural and spectroscopic data available on
Cd(II)-substituted human MT-3 indicated the
forma-tion of two metal thiolate clusters, similar to those
found in MT-1 and MT-2 Further investigation of
that protein pointed towards a highly dynamic
struc-ture [8] Recently, a high-resolution solution strucstruc-ture
of the C-terminal a-domain has become available The
data revealed a tertiary fold very similar to that of
MT-1 and MT-2, except for a loop that contains an
acidic insertion that is highly conserved in these
iso-forms The structure of the b-domain has escaped its
experimental solution, as no characteristic signals
attributable to its residues were observed using NMR
On the basis of homology modelling, a backbone
arrangement virtually identical to the corresponding domains in MT-1 and MT-2 was suggested [21] Despite the large number of structural data available for the MT family, only the structures of two MTs containing Cu(I) were known until now One of them
is the aforementioned yeast MT whose structure was successfully determined using both 2D NMR and X-ray diffraction [22–24] This protein forms one single domain that harbours eight Cu(I) ions Six of them are coordinated by three thiolate sulphur atoms, whereas a linear binding mode was observed for the remaining two The solution structure of N crassa MT backbone,
in which, like yeast, the MT solely binds Cu(I), repre-sents the second known structure of a copper thionein [25] Its polypeptide chain wraps around the copper sulphur cluster in a left-handed form in the N-terminal half of the molecule and in a right-handed form in the C-terminal half Due to the lack of copper isotopes with NMR-active spin ½, no metal–cysteine restraints were available to solve the positions of Cu(I) within the N crassa MT polypeptide fold
At present, the structural information on Cu(I)-loa-ded forms of mammalian MTs is rather limited
In vitro, Cu(I) titrations of isolated MT-2 and its sep-arate domains demonstrated that up to six Cu(I) ions can bind to each domain [26] In another extensive ti-tration study, it was postulated that zinc was required for the in vivo and in vitro folding of the two domains
of copper MTs [27] Replacement of Zn(II) by Cu(I) led to the proposal of the formation of Cu,Zn-MT intermediates and that, during the last steps of copper titration, the two domains merge into one However, earlier Cu(I) titration studies of rat liver MT clearly showed that the two domains remained separated [26] Additionally, the cooperative formation of (Cu3Zn2)a(Cu4Zn1)bMT)1 upon addition of Cu(I) to (Zn4)a(Cu4Zn1)bMT)1 indicated that the preference of Cu(I) for binding to the b-domain is only partial and not absolute, as widely accepted until now [27]
It was of interest to shed some light on the changes
of the molecular architecture of the two domains of vertebrate MT when Cu(I) is added to them For this task, the synthetic murine aMT-1 and bMT-1 domains were prepared for subsequent Cu(I) titrations Employ-ing NMR, we obtained an interestEmploy-ing and unexpected picture of the Cu(I) binding to the two single domains
Results and Discussion
Cu(I) titration of Zn4aMT-1 and Zn3bMT-1
As both the structure of native Zn7MT-1 was known, and several Cu(I) binding stoichiometries for its two
Trang 3domains had been proposed, it was of interest to shed
some more light upon their reactivity towards the
pres-ence of Cu(I) To this end, a Cu(I) titration study of
the two separated domains was performed employing
the combined detection of luminescence, circular
dichroism and1H NMR Solid-phase peptide synthesis
was successfully employed to prepare the independent
a- (residues 31–61) and b-domains (residues 1–30) of
murine MT-1 Either domain was fully reconstituted
under anaerobic conditions with Zn(II) to yield
Zn4aMT-1 and Zn3bMT-1 For each Cu(I) titration
step, a new sample was prepared in order to minimize
the risk of oxidation during sample manipulation
and measurement The Zn4aMT-1 and Zn3bMT-1
derivatives were separately titrated with Cu(I) under
a nitrogen atmosphere to yield Cu(I)–polypeptide
stoichiometries from zero to six The sample solution
contained 20% (v⁄ v) acetonitrile, as the presence of
soft ligands prevents Cu(I) from disproportionation to
Cu(II) and Cu(0)
CD and luminescence emission was measured in
order to assess the sample preparation quality and to
compare the obtained results with those previously
published [26,27] These physicochemical properties are
exclusively attributable to the metal-thiolate
chro-mophores that have been proven to be essential for the
proper polypeptide folding in other MTs [2] The
over-all shape of the CD spectra was essentiover-ally the same as
reported before (Fig 1) During the titration of the
a-domain, two positive dichroic bands developed at 248
and 300 nm, respectively, and one negative band at
275 nm The addition of Cu(I) to Zn3bMT-1 shifted
the positive dichroic band at 248 to 260 nm A second positive band at 300 nm, that was not present in the spectrum of Zn3bMT-1, appeared on addition of Cu(I) As in the case of the CD spectra, the results of luminescence emission were comparable to earlier stud-ies (Fig 2) An almost linear increase of intensity was observed until the addition of the third and fourth Cu(I) ions to the a- and b-domain, respectively Fur-ther Cu(I) addition led to a much more pronounced increase of intensity in both species
Two-dimensional 1H-1H NOESY spectra of each sample were acquired at 700 MHz (Figs 3 and 4) The spectrum of Zn4aMT-1, corresponding to the starting point of the aMT-1 titration, was consistent with a well-folded polypeptide Spin systems of the amide protons spread from 6.8 to 9.2 p.p.m Upon addition
of the first equivalent Cu(I), the spin systems of the starting point remained preserved, but additional new spin systems started to appear In the spectra of the samples containing two, three and four equivalents of Cu(I), these new spin systems were prevalent with the most and strongest signals observed for the three Cu(I)-containing sample On further additions of Cu(I), the signals faded away such that the spectra
of the six and seven Cu(I) titration steps were devoid
of cross-peaks (not shown)
For the b-domain similar results were obtained with the difference that the first addition of Cu(I) led only
to the reduction of signals in the NOESY spectrum and that new spin systems appeared only after the sec-ond equivalent Cu(I) was added The spectra of the samples containing three, four and five equivalents
-30 -20 -10 0 10 20
/ nm
Zn4- -MT + 1 eq Cu(I) + 2 eq Cu(I) + 3 eq Cu(I) + 4 eq Cu(I) + 5 eq Cu(I) + 6 eq Cu(I)
A
/ nm
Zn3- -MT + 1 eq Cu(I) + 2 eq Cu(I) + 3 eq Cu(I) + 4 eq Cu(I) + 5 eq Cu(I) + 6 eq Cu(I)
B
Fig 1 CD spectra of Zn4aMT (A) and
Zn3bMT (B) along the titration with Cu(I).
Samples containing 35 l M of the respective
domains dissolved in 15% (v⁄ v) acetonitrile,
20 m M sodium phosphate buffer pH 7.6
were prepared under nitrogen containing
<1 p.p.m O2.
Trang 4Cu(I) contained the same new spin systems The most
and strongest NOEs were observed in the spectrum
of the four Cu(I)-containing sample As with the
a-domain, progressive disappearance of NOEs without
reappearance of any new signals was the result of
Cu(I) to polypeptide stoichiometries higher than five
Taken together, the initial additions of Cu(I) to each
domain caused the disappearance of a large set of
NOESY cross-peaks and the parallel appearance of
another set of cross-peaks, until a clean 2D spectrum
belonging to a single species was obtained Judging
from the highest number of NOEs and the strongest
signals in the respective NOESY spectra, the recovery
of a single species was completed after the addition of
three Cu(I) equivalents to Zn4aMT-1 and of four
Cu(I) equivalents to Zn3bMT-1 This result was
surpri-sing insofar as structurally defined Cu(I)-containing
species were identified with such unexpectedly low
stoi-chiometries of Cu(I) to polypeptide Several different
Cu(I) binding stoichiometries had been proposed for
the two domains, among which Cu3aMT-1 and
Cu4bMT-1 had mostly been considered to be transient
intermediates in the pathways describing the formation
of the fully loaded domains [27–30] Cu6aMT-1 and
Cu6bMT-1 had been the most prominent among the
candidates for the fully Cu(I) loaded domains [26] In
the present titration study, however, the distinct
struc-tures disappear upon addition of more than three and
four Cu(I) equivalents without any sign of newly
form-ing defined structures We can only speculate what
happens at this stage of titration One possible
explan-ation for the disappearance of NOESY signals at high
Cu(I) concentration might be that two or more Cu(I)
binding modes coexist in an intermediate exchange regime, such that signals are exchange broadened and become invisible Of course, there is still the possibility that the separated domains are simply incapable of binding more than three and four Cu(I) without aggre-gating and denaturing, whereas in the native MT-1, the presence of the other domain would help to accommodate additional ions We do not believe that this is very likely, however, because of the similarity of the Zn4aMT-1 and Zn3bMT-1 structures with the structures of the domains of the intact protein (see below) [Correction added after publication 30 March 2007: in the preceding sentence the first term,
Zn3aMT-1 was corrected to Zn4aMT-1] The biophysi-cal similarities of intact protein and separated domains would also argue against this proposal [26]
Luminescence titration series also provide notewor-thy information Luminescence intensities increased almost linearly until Cu(I) stoichiometries of three and four for the a- and b-domain, respectively At this point, the curves were bent and further Cu(I) equiva-lents caused a much stronger, but also linear increase
of intensity As luminescence intensity is a measure of how the Cu-thiolate luminophore is shielded from solvent quenching, the titrations indicate that the shielding of the metal-thiolate cluster in the newly identified structures is not optimal compared with the situation with higher Cu(I):polypeptide stoichiometries
An explanation for this and the loss of structural information might be the formation of polymolecular structurally undefined aggregates of native MT or its single domains when they are overloaded with Cu(I) in the presence of unphysiologically high Cu(I)
0
10
20
30
40
50
60
/ nm
0 equivalents Cu(I)
6 equivalents Cu(I)
/ nm
0 equivalents Cu(I)
6 equivalents Cu(I)
mole equiv of Cu(I)
0 1 2 3 4 5 6
0 1 2 3 4 5 6 0
1000 2000 3000 4000 5000
0 1000 2000 3000 4000 5000
6000 7000
mole equiv of Cu(I)
Fig 2 Luminescence emission spectra of
Zn 4 aMT (A) and Zn 3 bMT (B) along the titra-tion with Cu(I) Samples containing 14 l M of the respective domains dissolved in 15% (v ⁄ v) acetonitrile, 20 m M sodium phosphate buffer, pH 7.6, were prepared under nitro-gen containing <1 p.p.m O2 Spectra were recorded at 25 C using slits of 15 and
20 mm for excitation and emission mono-chromators, respectively Excitation was at
k ¼ 300 nm The insets show the emission intensities plotted against the respective polypeptide stoichiometries.
Trang 5tions Unlike the observed distinct stoichiometries of
three and four Cu(I) leading to a sharp rise of the
luminescence, only a very small dependency was seen
in the circular dichroic measurements This was
also shown earlier by Bofill et al [27], although CD
spectrometry is obviously not sensitive enough to
detect comparable significant changes as deduced from
luminescence data
1H NMR and solution structures of Zn4aMT-1 and
Zn3bMT-1 From previous different studies on vertebrate Zn(II)-and Cd(II)-containing MTs, it was already known that the two domains form independently from each other and do not interact with each other Therefore, it was suggested that the two single domains possess very
Fig 3 Upper-left parts of the1H-1H NOESY
spectra of Zn4aMT (A), Zn4aMT +1 Cu(I) (B),
Zn 4 aMT +2 Cu(I) (C), Zn 4 aMT +3 Cu(I) (D),
Zn 4 aMT +4 Cu(I) (E) and Zn 4 aMT +5 Cu(I)
(F) All samples contained 1 m M polypeptide
dissolved in 15 m M acetate-d3, 15%
aceto-nitrile-d 3 , 10% D 2 O, 50 m M potassium
phos-phate, pH 6.5 and were prepared under a
nitrogen atmosphere containing less than
1 p.p.m O 2 Measurements were
per-formed at 283 K on a Bruker AVANCE 700
spectrometer operating at 700.13 MHz
using 600 ms mixing time.
Trang 6similar structures, if not identical, to their structure in
the intact protein Analysis of the NOESY and
TOC-SY (not shown) spectra of the two domains permitted
the full sequence-specific assignments, the identification
of the spin systems and the assignment of 398 and 252
of the NOESY cross-peaks of the a- and b-domain,
respectively The comparison of the chemical shifts
with those reported for the cadmium derivative
revealed very close similarities (supplementary Tables
S1 and S2) In the spectrum of the a-domain, the
reso-nances were shifted marginally by some hundredths of
a p.p.m The differences observed for the b-domain were more pronounced, with some deviations of up to 0.2 p.p.m., which is probably due to an increased flexi-bility in this domain The spin system patterns repor-ted for the published cadmium protein, however, were preserved in both domains Most importantly, 22 of the 28 long-range NOEs that were reported for
Cd7MT-1 were also found in the spectra of the zinc-containing a- and b-domain (Table 1) It should be noted that three of the six missing long-range NOEs were assigned to residues of the linker region between
Fig 4 Upper-left parts of the 1 H- 1 H-NOESY spectra of Zn 3 bMT (A), Zn 3 bMT +1 Cu(I) (B),
Zn3bMT +2 Cu(I) (C), Zn 3 bMT +3 Cu(I) (D),
Zn3bMT +4 Cu(I) (E), and Zn3bMT +5 Cu(I) (F) Sample and measurement conditions were the same as described in Fig 3.
Trang 7the two domains Therefore, the lack of the second
domain seems to lead to increased flexibility at either
end that would build up the linker region in native
MT-1 The fact that the majority of the long-range
NOEs observed in Cd7MT-1 is still present in the
sin-gle zinc-containing domains suggests that the global
structures of the two domains are preserved, regardless
of whether zinc or cadmium is bound to them and also
regardless of the existence of the second domain
The assigned peaks of the a-domain were integrated
and their consistency with the published solution
struc-ture was checked using the program dyana and the
published solution structure of the respective
cad-mium-containing domain of the intact protein as a
starting point The resulting structure family had a
tar-get function of 0.15 ± 0.11 A˚2 and rmsd values of
1.51 ± 0.27 A˚ and 2.22 ± 0.30 A˚ with respect to the
mean structure for the backbone and all heavy atoms,
respectively In the previous study on the Cd7
deriv-ative, metal–sulphur connectivities were obtained using
the NMR-active cadmium isotope113Cd and added to
the structure calculation process Using these connec-tivities together with the data for Zn4aMT-1 resulted
in a target function of 0.52 ± 0.13 A˚2and rmsd values
of 1.04 ± 0.12 A˚ and 1.51 ± 0.18 A˚ for a new struc-ture family Both mean strucstruc-tures were essentially the same, with rmsd values of 0.79 A˚ and 1.19 A˚ for the backbone and heavy atoms, respectively Thus, the addition of metal–sulphur connectivities to the struc-ture calculation of Zn4aMT-1 resulted in a better-resolved structural family but did not change the overall protein fold Figure 5 shows the superposition
of the structural family obtained with these additional connectivities and the mean structure of the previously published Cd4aMT-1, showing that they possess the same structure, within the experimental uncertainty
A separate structure determination of Zn3bMT-1 on the basis of the present NMR data was not attempted,
as only a small number of NOEs and only four long-range NOEs were found in its NOESY spectrum Only with the help of the metal–sulphur connectivities dis-covered using the cadmium-containing derivative could
a structure determination have been possible How-ever, preserved chemical shifts and spin system pat-terns indicate an identical structure for Zn3bMT-1 as for Cd3bMT-1 As anticipated, the structures of the separated Zn(II)-containing domains are indistinguish-able from those of Cd7MT-1 and, knowing the appear-ance of the starting points, it was of interest to know
to what extent they would change in the presence of copper(I)
1H NMR and solution structures of ZnxCu3aMT-1 and ZnyCu4bMT-1
The NOESY spectra of the ZnxCu3aMT-1 and
ZnyCu4bMT-1 domains are markedly different from the starting Zn4aMT-1 and Zn3bMT-1 derivatives, pointing to a different arrangement of the polypeptide chains, which is probably needed to accommodate the resulting Cu(I)- or mixed metal–sulphur clusters (Fig 6) From a cursory inspection of the superim-posed spectra, it has already become clear that the addition of Cu(I) not only leads to a completely differ-ent pattern of spin systems, but also to a significantly higher number of NOEs Therefore, we expected the structures of the newly identified Cu(I)-containing domains to be more rigid and distinct from their Zn(II)-containing forms The spectra of both the only Zn(II)-containing b-domain and its Cu(I)4 derivative seem to be of lower quality with large areas of broad overlapping peaks This behaviour might be due to higher flexibility within the b-domain which has been reported already before [17–20] TOCSY spectra (not
Table 1 Comparison of the long-range NOEs (d ij j > I +4) of Zn 4
-aMT and Zn 3 bMT with those observed for Cd 7 MT [20] Presence
(+) or absence (–) of NOEs is indicated.
b-Domain
a-Domain
Trang 8shown) were recorded for the ZnxCu3aMT-1 and
ZnyCu4bMT-1 derivatives and used, together with the
NOESY spectra, to obtain their sequence specific
assignments (supplementary Tables S1 and S2) The
resonances of the new Cu(I)-containing species differed
mostly by several tenths of a p.p.m from those of
their starting points, thereby confirming the
observa-tions mentioned above
In the NOESY spectrum of ZnxCu3aMT-1 502,
NOEs were assigned, integrated and converted into
distance constraints In the last dyana run, a set of
200 structures was generated out of which the 20 best
were combined to a structure family (Fig 7) The
tar-get function was 0.43 ± 0.10 A˚2, and the rmsd values
were 0.70 ± 0.12 A˚ and 1.03 ± 0.14 A˚ for the poly-peptide backbone and heavy atoms, respectively Like-wise, 500 NOEs of the ZnyCu4bMT-1 spectrum were used to derive a structure family for this species (Fig 8) In this case, a target function of 0.19 ± 0.02 A˚2 and rmsd values of 0.49 ± 0.21 A˚ and 0.72 ± 0.21 A˚ for the backbone and heavy atoms were obtained
As with other known structures of MTs, those pre-sented here also lack typical secondary structure ele-ments The structure of ZnxCu3aMT-1 is roughly a two-level structure (Fig 7) where the segment 5–10 of the a-domain polypeptide backbone forms the first level The stretch 10–16 links the first with the second
Fig 5 Superposition of the present family of 30 structures of Zn4aMT (blue) with the published average structure of Cd4aMT (red) The coordinates of the Cd 4 aMT structure were extracted from the Brookhaven protein data bank (1DFS) In the last run of structure calculations for Zn 4 aMT 398 upper distance limits (upl) obtained from the assignment of its NOESY spectrum and the metal-sulphur connectivities repor-ted by Zangger et al [20] for Cd4aMT were used as input for the program DYANA Twenty out of 200 structures were combined into a struc-ture family with a target function of 0.52 ± 0.13 A˚2 and RMSD values of 1.04 ± 0.12 A ˚ and 1.51 ± 0.18 A˚ for the backbone and all heavy atoms, respectively.
Fig 6 1 H- 1 H-NOESY spectra of Zn4aMT (red) superimposed with that of Zn x Cu 3 aMT (green) (A) and of Zn 3 bMT (red) superim-posed with that of ZnyCu4bMT (green) (B) Sample and measurement conditions were the same as described in Fig 3.
Trang 9level, which is constituted by the second half of the
polypeptide chain The region between residue 20 and
26 includes a loop that is more disordered than the
rest of this domain, shielding the rear of its core The
positions of several cysteine sulphur atoms are not
very well defined (Fig 7) Nevertheless, the 11
cyste-ines seem to form a somewhat broader cavity than in
its Cd4aMT-1 counterpart, with the subgroup of Cys3,
11 and 18 being rather isolated from the other eight
cysteines
The polypeptide backbone of the b-domain wraps
around its core in a right-handed fashion (Fig 8) The
central part of its polypeptide chain, residues 8–24,
limits an almost elliptical planar structure At the point
where the two ends of the polypeptide chain would
meet, they leave the central plane and continue in
opposite directions Again, outside the uncertainty in
the positions of some of the sulphur atoms, the cavity
encased by the cysteines is somewhat broader than its
Cd3bMT-1 counterpart, although in this case the nine
cysteines still point to a unique core
The superposition of the newly identified Cu(I)-con-taining domains onto the mean structures of their Cd(II)-coordinating forms highlights the striking struc-tural differences that are caused only by the binding
of different metal ions to the two domains of MT (Fig 9) When the structures are superimposed throughout the full length of their polypeptide chains only very faint similarities of some parts of the poly-peptide backbone folds could be observed Separate superpositions of stretches 3–12, 11–20 and 18–28 were also attempted For both domains, the first and third stretches gave very poor overlap, while the central part showed a more pronounced similarity This could indi-cate that each domain adapts to host the additional copper(I) ions by opening up and rearranging its N- and C-terminal parts, minimizing the structural perturbation of its central part
The arrangement of the cysteine sulphur donor atoms within the two Cu(I)-containing domains is also shown in Figs 7 and 8 Although, from the present data, it is neither possible to guess the number of
Fig 7 Stereoview of the 20-structure family
of Zn x Cu 3 aMT Polypeptide backbone bonds
are shown in grey, cysteinyl side chain
bonds in blue and sulphur atoms as yellow
spheres 502 NOEs were converted into upl
and were used as input for the structure
calculation program DYANA Twenty out of
200 structures were combined into a
struc-ture family with a target function of
0.43 ± 0.10 A˚2 and RMSD values of
0.70 ± 0.12 A ˚ and 1.03 ± 0.14 A˚ for the
backbone and all heavy atoms, respectively.
Fig 8 Stereoview on the family of 20 best
structures of Zn y Cu 4 bMT Polypeptide
back-bone bonds are shown in grey, cysteinyl
side chain bonds in blue and sulphur atoms
as yellow spheres 500 NOEs were
conver-ted into upl and were used as input for the
structure calculation program DYANA Twenty
out of 200 structures were combined into a
structure family with a target function of
0.19 ± 0.02 A ˚ 2 and rmsd values of
0.49 ± 0.21 A ˚ and 0.72 ± 0.21 A˚ for the
backbone and all heavy atoms, respectively.
Trang 10residual Zn(II) ions in each structure nor the overall
topology of the clusters, it appears that all cysteine
res-idues point towards the inside of the respective
domain, as expected if all of them are to be involved
in metal coordination In turn, the somewhat broader
cavities encased by the cysteines are consistent with the
increased number of metals in each domain
At this point it was still an open question as to
whether or not the single species ZnxCu3aMT-1 and
ZnyCu4bMT-1 would be stable when the other
Cu(I)-containing domain was also present in solution To this
end, both Cu(I)-containing domains were prepared as
for the titration experiment mentioned above,
com-bined in equal amounts at a final concentration of
1 mm each and incubated for >48 h before the
meas-urement of their1H NMR spectra The observed
NO-ESY and TOCSY spectra (not shown) consisted of the
sum of the respective spectra of the single species The
spectral resonances were assigned to all the protons
present in the two domains and are listed in
supple-mentary Tables S1 and S2 This result indicates that
the two domains are stable and independent from each
other Cu(I) is not transferred between the two domains
to form new species with higher and lower
Cu(I):poly-peptide stoichiometries As no additional NOEs and⁄ or changes of the spectral resonances were observed in the NOESY spectrum of the mixture, an interaction of the two single domains and the formation of one single Cu(I)-containing domain with the involvement of both domains could be excluded for the present
Zn(x+y)Cu7MT-1 stoichiometry
Conclusion
The Cu(I) titration of the independent Zn(II)-loaded domains of mouse MT-1 revealed Cu(I) stoichiometries
of three and four for the a- and b-domain, respectively The presence of Cu(I) led to dramatic conformational changes of both polypeptide folds Cu(I) stoichio-metries of up to six Cu(I) ions each led to the progressive disappearance of the altered structures [Correction added after publication 30 March 2007: in the preced-ing sentence, disappearpreced-ing of the affered structurer, was corrected to disappearance of the altered structures] Unfortunately, due to the lack of metal Æ sulphur constraints, the Cu(I) positions within the resolved polypeptide folds remained unclear Therefore, crystal-lization of the newly identified Cu(I)-containing species
Fig 9 (A) Stereoview of the superposition
of the Cd 4 aMT-1 mean structure (red) to the structure family of ZnxCu3aMT-1 (blue) (B) Stereoview of the superposition of the
Cd 3 bMT-1 mean structure (red) to the struc-ture family of ZnyCu4bMT-1 (blue).